The experiments proposed in this application are designed to investigate the mechanism of growth hormone action. To achieve this objective, growth hormone responsive hepatic messenger RNA sequences will be defined and the coordinate regulation of these mRNA sequences by growth hormone and other hormones will be determined. In addition, the question of whether the changes induced by growth hormone on specific mRNA sequences are the result of increased transcription and/or stabilization of the mRNA sequence will be examined. Two approaches will be used to measure tissue levels of specific mRNA sequences. As an initial screening technique for growth hormone responsive mRNA sequences, total mRNA will be isolated from livers of in vivo treated animals or from cultured hepatocytes. The mRNA will be used to direct protein synthesis in the cell free rabbit reticulocyte lysate translational system in the presence of isotopically labeled amino acids and the labeled proteins will be separated by two-dimensional gel electrophoresis. Individual spot intensity which is a reflection of the quantity of functional mRNA coding for that spot will be assessed by video imaging. To confirm the results of the in vitro translational system and to further study the mRNA sequences of interest, complementary DNA probes will be made for the growth hormone responsive sequences. Recombinant plasmids will be prepared using hepatic poly(A)-containing RNA as starting material for the complementary DNA inserts. Transformed colonies will be screened by colony hybridization with [32P] cDNA prepared from poly(A) +RNA obtained either from thyroidectomized or growth hormone treated thyroidectomized animals. Plasmid DNA from colonies which yield differential signals will be used for hybrid selected translation and the in vitro synthesized product identified by gel electrophoresis. Appropriate cDNA probes to the growth hormone responsive mRNA sequences will be used to measure rate of synthesis of that mRNA in hepatocyte cultures and in nuclei obtained from control and growth hormone treated tissue. By similar techniques the rate of degradation of specific mRNA will also be examined. These studies will provide significant insight on the mechanism of growth hormone action at a pretranslational level and provide the foundation for experiments designed to examine the link between hormone-plasma membrane receptor interaction and pretranslational events. In addition, insight into the full complexity of disorders of growth in man will be gained.